The use of multi-drug regimens which target different HIV encoded proteins has dramatically changed the course of AIDS. The concept of highly active retroviral therapy (HAART) is to increase antiviral potency while minimizing the probability of mutant viral resistance. Despite the impact on AIDS, there are complications of HAART, including emerging resistant mutants, toxicities of HAART with other medications, and the long term cost of a lifetime of medication. An alternative or possible adjuvant to HAART therapy is the use of combinatorial gene therapy modification of hematopoietic cells. Research from this program has led to the development of a lentiviral vector harboring three different therapeutic genes targeting different steps in the HIV life cycle. This triple RNA inhibitor combination has proven to be the most potent of our tested anti-HIV approaches to date. This vector will enter a phase I clinical trial for hematopoietic stem cell transduction (HSC) of AIDS/lymphoma patient cells sometime in late 2006, and T-lymphocyte trials in 2007. The present proposal capitalizes upon the findings of the previous funding period, and proposes to develop new anti-HIV combinatorial RNAs with enhanced potency and safety to prevent or minimize the emergence of viral escape mutants. The proposed studies will explore a novel polycistronic micro RNA system as a possible platform for co-expressing combinations of anti-HIV siRNAs and nucleolar localizing inhibitory RNAs. The proposed studies incorporate novel anti-HIV targeting approaches which will be tested alone and in combinatorial fashion with already established inhibitory RNAs. Delivery of the inhibitory genes will be via lentiviral vector transduction of the combinatorial constructs into HSCs for in vitro and in vivo evaluations of anti-HIV efficacy and potential toxicities. The proposed studies will allow us to critically test the hypothesis that combinatorial gene therapy for treatment of HIV infection can be effected via genetically modified HSC transplantation. The in vivo testing will be conducted using transduced HSCs and bone marrow transplantation in the pigtail macaque (M. nemestrina) as part of a collaborative effort with Drs Kiem and Hu through the University of Washington Primate Center. The safety and efficacy of the constructs will be critically evaluated in this system. The Specific Aims of this study are: 1) Development of new strategies for multiplexing anti-HIV gene expression;2) Expression strategies and mechanism of action studies; 3) Evaluation of in vivo hematopoietic repopulation capabilities and resistance to viral challenge of vector transduced macaque CD34+ hematopoietic progenitor cells. These studies will provide the first detailed, in vivo investigations of antiviral RNAi and other inhibitory RNA combinations in a primate HSC gene therapy setting, and should provide the type of safety and efficacy data required to bring the lead combinatorial constructs to human clinical trials. [unreadable] [unreadable] [unreadable] [unreadable]